Cell growth is highly regulated in normal tissues by a variety of mechanisms in order to guide normal tissue development and homeostasis. A cell's response to the “microenvironment” is a major portion of the growth regulatory machinery. The microenvironment consists of soluble factors, adjacent cell surfaces and molecules of the extracellular matrix (ECM). Information within the microenvironment is primarily detected by cell surface receptors that bind specific molecules found in the microenvironment and elicit varied cell responses for growth, morphogenesis or differentiation.
The work reported here focuses on cell interactions with the ECM in general and a specialized form of ECM, called the basement membrane (BM). This specialized extracellular matrix serves not only as a barrier between cell layers, but also as an active signaling substrate that regulates epithelial cell growth, differentiation and tissue architecture. Key signaling components of the BM are the laminin glycoproteins. Laminin-1 alone can induce cell shape changes, growth arrest, and functional differentiation when added to cultured mammary epithelial cells (MECs). Signals from laminin are mediated by direct binding to multiple cell-surface receptors whose individual functions are not completely defined. It has been hypothesized that the aberrant behavior of tumor cells arises, in part, from alterations in cell-BM interactions. In support of this model, tumor cells frequently demonstrate altered responsiveness to BM proteins, indicating changes in BM receptor functions. Significantly, the laboratory of Dr. Mina Bissell has demonstrated that functionally normal MECs can be distinguished from tumorigenic MECs by their growth characteristics when cultured within a 3-dimensional gel of BM proteins (3D-BM assay); functionally normal MECs cultured within Matrigel will grow from single cells to form multi-cellular, polarized acinar structures that arrest growth, whereas tumorigenic MECs grow as disorganized cell masses with unregulated cell growth. The 3-D basement membrane assays distinguish between normal and tumorigenic mammary epithelial cell behavior. Normal cells growth arrest as acinar structures, whereas tumor cells do not growth arrest. This tumor cell characteristic is referred to as a “tumorigenic phenotype”. This growth difference has been described in U.S. Pat. No. 5,846,536 incorporated by reference herein. Although it is evident that the cellular machinery that senses the BM is altered in tumorigenic epithelial cells, it is less certain where the critical changes occur. Studies of cell-BM interactions have largely focused on the integrins, an extensively characterized family of heterodimeric receptors. However, integrin signaling generally favors tumor cell growth and metastasis, and no integrin has been unambiguously assigned the role of tumor suppressor, leaving the possibility that other important receptors may still need to be investigated. The present invention relates to the characterization of one such receptor, dystroglycan (DG).
Accordingly, it is an object of the present invention to provide an assay of dystroglycan expression. This assay may be used to show that the laminin binding portion of α-dystroglycan is lost in tumor cells.
It is another aspect of the invention to provide an assay of dystroglycan proteolysis and shedding through the detection of cell-surface α-dystroglycan. This assay focuses on the relative ratio of α- to β-dystroglycan at the surface of cells, as compared to cells like the BT474 cells (FIG. 2, Lane 2) which shed little or no α-dystroglycan.
It is another aspect of the invention to provide an assay for the generation of dystroglycan fragments that can be used to assay for inhibitors of the metaloproteinase(s) cleaving and shedding α-dystroglycan.
It is yet another aspect of the invention to provide an assay that utilizes α-dystroglycan protein, or derivative thereof, as a substrate for a cell-free assay measuring the activity of the protease(s) cleaving it. The substrate consists of either the full-length α-dystroglycan molecule, a fragment thereof, or a synthetic peptide capable of being recognized and cleaved by the enzyme cleaving α-dystroglycan.